rfx2 Antibody

What is RFX2 and why is it important in research?

RFX2 (Regulatory Factor X 2) is a transcription factor that contains a highly-conserved winged helix DNA binding domain. It acts as a transcriptional activator that can bind DNA as either a monomer or heterodimer with other RFX family members . RFX2 is structurally related to regulatory factors X1, X3, X4, and X5, and plays crucial roles in several biological processes:

• Spermiogenesis and male fertility - RFX2-deficient male mice are completely sterile due to a developmental block in haploid cells

• Transcriptional regulation of cilia/flagella-related genes

• Tumor suppression in lung adenocarcinoma, where its expression is significantly downregulated compared to normal tissue

Research on RFX2 has significant implications for understanding developmental biology, reproductive medicine, and cancer pathology.

Which applications are most suitable for RFX2 antibodies?

Based on validation data across multiple commercial antibodies, RFX2 antibodies have been successfully used in the following applications:

The choice of application should be dictated by your specific research question, with consideration for species reactivity and epitope accessibility.

How do I select the appropriate RFX2 antibody for my experiment?

When selecting an RFX2 antibody, consider these critical factors:

• Epitope specificity: Different antibodies target distinct regions of RFX2. For example, some target N-terminal regions (AA 1-130) , while others target C-terminal regions or the DNA binding domain. The epitope location may affect antibody performance in specific applications.

• Species reactivity: Confirm cross-reactivity with your experimental model. Some antibodies are specifically validated for human samples , while others show reactivity with mouse and rat samples .

• Validation data: Review validation data for your intended application. For instance, antibody ABIN7150380 is validated for ELISA, IHC, and IF but not Western blotting .

• Clonality: Polyclonal antibodies may provide higher sensitivity but potentially lower specificity compared to monoclonals. Most commercially available RFX2 antibodies are polyclonal .

• Host species: Consider compatibility with other antibodies in multi-labeling experiments to avoid cross-reactivity.

For specialized applications like ChIP-Seq, select antibodies specifically validated for chromatin immunoprecipitation protocols .

What are the optimal protocols for using RFX2 antibodies in immunohistochemistry?

For optimal immunohistochemical detection of RFX2, follow this methodological approach:

• Tissue preparation:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Process and embed in paraffin following standard protocols

  • Section at 4-6 μm thickness

• Antigen retrieval:

  • Heat-induced epitope retrieval is recommended

  • Use citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Boil sections for 15-20 minutes followed by cooling at room temperature

• Blocking and antibody incubation:

  • Block endogenous peroxidase with 3% H₂O₂ in methanol

  • Apply protein block (e.g., 5% normal serum)

  • Incubate with primary RFX2 antibody at recommended dilutions (typically 1:200-1:500)

  • For human LUAD samples, higher sensitivity may be required as RFX2 is significantly downregulated in these tissues

• Detection system:

  • Use appropriate HRP-conjugated secondary antibody or detection kit

  • Develop with DAB substrate

  • Counterstain with hematoxylin

• Controls:

  • Include positive control tissues known to express RFX2 (testis tissue shows robust expression)

  • Include negative controls by omitting primary antibody

  • Consider RNAi validation experiments to confirm specificity

This protocol has been successfully employed to detect reduced RFX2 expression in LUAD tissues compared to adjacent normal tissues .

How can I optimize RFX2 antibodies for ChIP-Seq experiments?

ChIP-Seq with RFX2 antibodies requires specific optimization steps:

• Cell preparation:

  • For studying RFX2 in spermatogenesis, use dissociated germ cell preparations from P21 wild-type mice

  • For cancer studies, use appropriate cell lines (e.g., A-549 and NCI-H358 for LUAD research)

  • Crosslink cells with 1% formaldehyde for 10 minutes at room temperature

• Chromatin preparation:

  • Lyse cells and isolate nuclei

  • Sonicate chromatin to generate fragments of 200-500 bp

  • Verify fragment size by agarose gel electrophoresis

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate cleared chromatin with 2-5 μg RFX2 antibody overnight at 4°C

  • Add protein A/G beads and incubate for 1-3 hours

  • Wash beads thoroughly with increasing stringency buffers

• DNA purification and library preparation:

  • Reverse crosslinks and purify DNA

  • Prepare sequencing libraries following standard protocols

  • Include input control for normalization

• Data analysis considerations:

  • RFX2 binding peaks are often concentrated near transcription start sites (TSS)

  • Approximately 1/3 of peaks are located within promoter regions (-500 to +50)

  • Use motif analysis tools to identify the characteristic X-box motif bound by RFX2

This approach has successfully identified nearly 3,000 RFX2 binding sites genome-wide, providing insights into its gene regulatory functions .

What are the best conditions for storage and handling of RFX2 antibodies?

To maintain optimal antibody performance:

• Long-term storage:

  • Store at -20°C in manufacturer-provided buffer

  • Most RFX2 antibodies are provided in PBS with glycerol (typically 50%) and preservatives

  • Avoid repeated freeze-thaw cycles

• Working solutions:

  • Dilute in appropriate buffer immediately before use

  • For IHC/IF, use PBS with 1% BSA or commercial antibody diluent

  • For Western blotting, use TBST with 1-5% non-fat dry milk or BSA

• Stability considerations:

  • Most RFX2 antibodies remain stable for one year after shipment when stored properly

  • Aliquoting is generally unnecessary for small volume antibodies (e.g., 20 μL) stored at -20°C

  • Some formulations contain BSA (0.1%) as a stabilizer

• Preservatives and safety:

  • Many commercial preparations contain sodium azide (0.02-0.03%)

  • Handle with appropriate safety precautions

  • Avoid contamination by using sterile technique when handling antibody solutions

Follow manufacturer-specific recommendations, as buffer compositions may vary between suppliers.

How does RFX2 contribute to cancer biology, particularly in lung adenocarcinoma?

RFX2 plays a significant tumor-suppressive role in lung adenocarcinoma (LUAD) through several mechanisms:

• Expression patterns:

  • RFX2 is markedly downregulated in LUAD tissues compared to adjacent normal tissues

  • IHC staining has confirmed attenuated RFX2 expression in patient LUAD specimens

  • RFX2 is lowly expressed in LUAD cell lines including A-549, NCI-H358, Calu-3, and H1975

• Immune regulation:

  • RFX2 expression positively correlates with CD8+ T cell infiltration specifically in LUAD (not in lung squamous cell carcinoma)

  • RFX2 overexpression increases:

    • Infiltrating CD8+ T cells within transplanted tumors

    • Release of IFN-γ, GZMB, and PRF1 by CD8+ T cells

    • CD8+ T cell activation

  • RFX2 overexpression decreases PD-L1 expression, inhibiting immune escape

• Signaling pathway:

  • RFX2 directly activates RASSF1 transcription by binding to its promoter

  • This activation increases YAP phosphorylation in the Hippo pathway

  • RASSF1 knockdown reverses the inhibitory effects of RFX2 overexpression on immune escape

  • The RFX2-RASSF1-Hippo signaling axis represents a novel regulatory mechanism in LUAD

• Malignant phenotype:

  • RFX2 overexpression inhibits viability and invasion of LUAD cells

  • RFX2 overexpression enhances apoptosis of LUAD cells

These findings suggest RFX2 as a potential therapeutic target and prognostic marker in LUAD, with particular relevance to immunotherapy approaches.

What is the role of RFX2 in spermatogenesis and male fertility?

RFX2 is a key regulator of spermiogenesis through several critical functions:

• Developmental expression pattern:

  • RFX2 expression increases during postnatal testis development

  • Highest expression occurs in spermatocytes and early spermatids

• Fertility impact:

  • Male RFX2-deficient mice are completely sterile

  • Spermatogenesis progresses through meiosis in these mice, but haploid cells undergo a complete developmental block

• Cellular mechanisms:

  • RFX2 knockout leads to increased germ cell apoptosis, particularly evident at postnatal days P21 and P28

  • Apoptotic multinucleate giant cells appear in RFX2-deficient testes

  • Acrosomal formation begins to fail in the absence of RFX2

• Gene regulation:

  • ChIP-Seq analysis has identified nearly 3,000 RFX2 binding sites genome-wide

  • About 1/3 of these binding sites are located in promoter regions, with peaks concentrated near transcription start sites

  • RFX2 regulates numerous testis-specific genes and cilia/flagella-related genes

  • RFX2 binding sites contain a characteristic X-box motif

• Molecular targeting:

  • RFX2 knockout was achieved by replacing exons 6 and 7, which encode the DNA binding domain

  • This deletion results in a frameshift and premature termination of protein translation

This evidence establishes RFX2 as an essential transcription factor for male fertility, with potential implications for diagnosing and treating certain forms of male infertility.

How can RFX2 antibodies be used to investigate transcriptional networks?

RFX2 antibodies enable sophisticated investigations of transcriptional networks through several advanced approaches:

• ChIP-Seq analysis:

  • RFX2 antibodies can identify genome-wide binding sites

  • Analysis revealed ~3,000 statistically significant RFX2 binding peaks

  • Distribution analysis showed that ~1/3 of peaks are in promoter regions (-500 to +50)

  • The most robust peaks (lowest p-value) are preferentially enriched in promoter regions

  • Peaks show marked concentration near transcription start sites (TSS)

• Motif analysis integration:

  • After ChIP-Seq with RFX2 antibodies, recovered sequences can be analyzed for enriched motifs

  • RFX2 recognizes a characteristic X-box motif that can be compared with consensus motifs in databases like JASPAR

  • Motif analysis helps distinguish direct RFX2 targets from indirect regulatory effects

• Multi-omics approaches:

  • Combine RFX2 ChIP-Seq with RNA-Seq from RFX2 knockout or overexpression models

  • This integration identifies which binding events lead to functional changes in gene expression

  • In LUAD research, RFX2 overexpression followed by transcriptomic analysis revealed effects on immune-related genes and RASSF1 expression

• Protein-protein interaction studies:

  • RFX2 antibodies can be used for co-immunoprecipitation to identify protein complexes

  • RFX2 can bind DNA as a monomer or heterodimer with other RFX family members

  • Identifying these interactions helps map the complete regulatory network

• Pathway validation experiments:

  • After identifying potential regulatory pathways (e.g., RFX2-RASSF1-Hippo signaling), antibodies can validate protein expression changes

  • For example, RFX2 overexpression increases YAP phosphorylation through RASSF1 activation

These methodologies provide complementary perspectives on RFX2-mediated transcriptional regulation, enabling construction of comprehensive gene regulatory networks.

Why might I observe differences in RFX2 detection between tissues and cell lines?

Variability in RFX2 detection can result from several factors:

• Tissue-specific expression levels:

  • RFX2 expression varies significantly between tissues

  • Highest expression is observed in testis, particularly in spermatocytes and early spermatids

  • Expression is markedly reduced in LUAD compared to normal lung tissue

  • These natural variations may lead to different detection sensitivities

• Epitope accessibility issues:

  • Different antibodies target distinct regions of RFX2 (e.g., AA 1-130, AA 323-429, C-terminal)

  • Protein interactions or conformational changes may mask specific epitopes

  • Fixation methods can differentially affect epitope accessibility

  • For difficult samples, try antibodies targeting different RFX2 regions

• Technical considerations:

  • Fixation time affects antigen preservation (over-fixation can reduce signal)

  • Antigen retrieval methods may need optimization for specific tissues

  • For IHC in LUAD tissues, where RFX2 is downregulated, more sensitive detection systems may be required

• Post-translational modifications:

  • RFX2 may undergo tissue-specific modifications affecting antibody recognition

  • Consider using antibodies specifically designed to recognize unmodified forms

• Experimental validation:

  • Include positive controls from tissues known to express high RFX2 levels (e.g., testis)

  • Use RFX2-overexpressing cells as positive controls for low-expressing samples

  • Consider orthogonal validation methods (e.g., mRNA detection)

Understanding these factors can help troubleshoot unexpected results and design appropriate experimental controls.

How can I validate RFX2 antibody specificity for my research application?

Rigorous validation of RFX2 antibody specificity is essential for reliable research outcomes:

• Genetic validation approaches:

  • Test antibody in RFX2 knockout models - complete absence of signal confirms specificity

  • Use RFX2-deficient mouse models where exons 6-7 are deleted

  • Employ siRNA or shRNA knockdown and confirm reduced signal intensity

• Overexpression controls:

  • Test antibody in cells overexpressing RFX2 and verify increased signal

  • Use lentiviral infection approaches for stable RFX2 overexpression

  • Include tagged RFX2 constructs and perform parallel detection with anti-tag antibodies

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide (e.g., AA 1-130 for ABIN7150380)

  • Specific signals should be significantly reduced or eliminated

  • Non-specific signals will persist despite peptide competition

• Multiple antibody validation:

  • Test multiple antibodies targeting different RFX2 regions

  • Concordant results with antibodies recognizing distinct epitopes support specificity

  • Compare antibodies targeting N-terminal regions (AA 1-130) versus C-terminal regions

• Cross-reactivity assessment:

  • Test reactivity against other RFX family members with similar domains

  • RFX2 is structurally related to RFX1, RFX3, RFX4, and RFX5

  • Western blotting should show a single band at the expected molecular weight (~80 kDa)

These validation approaches should be documented and reported in publications to establish confidence in experimental findings.

What factors should I consider when designing experiments to study RFX2 in disease models?

When investigating RFX2 in disease models, consider these critical experimental design factors:

• Model selection considerations:

  • For cancer studies:

    • A-549 and NCI-H358 cell lines show the lowest RFX2 expression among LUAD cell lines

    • In vivo tumor models with RFX2 manipulation allow study of immune cell infiltration

  • For fertility studies:

    • RFX2 knockout mice provide a complete sterility model

    • Staged postnatal development studies (P14, P21, P28, P60) capture critical transitions

• Experimental timing:

  • RFX2 functions are developmentally regulated

  • In spermatogenesis, phenotypes become apparent at specific stages

  • Apoptotic effects in RFX2-deficient testes increase progressively (2× at P21, 8× at P28)

  • Design time-course experiments to capture dynamic effects

• Manipulation approaches:

  • Genetic models:

    • Targeted deletion of exons 6-7 (DNA binding domain) creates functional knockout

    • Lentiviral overexpression allows rescue experiments

  • Pharmacological considerations:

    • When studying the Hippo pathway downstream of RFX2, consider YAP inhibitors like PY-60

    • Small molecule modulators can help dissect pathway components

• Readout selection:

  • Cellular endpoints:

    • Apoptosis (TUNEL assay for RFX2-deficient testes)

    • Proliferation (CCK-8 assay for cancer cells)

    • Invasion (Transwell assay)

  • Molecular endpoints:

    • RASSF1 expression and YAP phosphorylation status

    • IFN-γ, GZMB, PRF1 release by CD8+ T cells

    • PD-L1 expression in cancer cells

• Translational relevance:

  • For cancer studies, correlate findings with patient data

  • IHC analysis of patient specimens can validate expression patterns

  • Consider clinical parameters like immune cell infiltration, which correlates with RFX2 expression

Careful consideration of these factors will strengthen experimental design and enhance the translational impact of RFX2 research.

How do the current generation of RFX2 antibodies compare with emerging antibody technologies?

Current RFX2 antibodies and emerging technologies offer different advantages for research applications:

• Current generation RFX2 antibodies:

  • Predominantly polyclonal antibodies from rabbit or mouse hosts

  • Available in various formats: unconjugated, HRP-conjugated, FITC-conjugated, biotin-conjugated

  • Typically generated against specific protein regions (e.g., AA 1-130, AA 323-429)

  • Purification methods include protein G affinity purification (>95% purity)

  • Applications validated include ELISA, IHC, IF, WB, and IP

• Emerging antibody technologies comparison:

  • De novo designed antibodies:

    • New approaches like RFdiffusion enable atomically accurate antibody design

    • These methods fine-tune models on antibody structures and use specialized design networks

    • Potential advantages include higher specificity and reduced cross-reactivity

    • Currently no reports of de novo designed RFX2 antibodies, but technology is applicable

  • Enhanced validation approaches:

    • Orthogonal RNAseq validation verifies antibody specificity through correlation with transcript levels

    • This approach provides stronger evidence for antibody specificity than traditional methods

  • AI-driven prediction tools:

    • Models like AlphaFold3 can predict antibody-antigen structures with high accuracy

    • These tools could help screen candidate RFX2 antibodies in silico before experimental validation

    • Currently used to filter designs that would have "incorrect epitope targeting"

• Comparative performance metrics:

While emerging technologies show promise for next-generation RFX2 antibodies with improved properties, current commercially available antibodies remain essential tools for RFX2 research when properly validated and optimized.

What are the most promising research directions involving RFX2 antibodies?

Based on current literature and technological developments, several promising research directions utilizing RFX2 antibodies emerge:

• Cancer immunotherapy applications:

  • RFX2's role in regulating CD8+ T cell infiltration and PD-L1 expression in LUAD suggests therapeutic potential

  • RFX2 antibodies could help identify patients likely to respond to immunotherapy through IHC screening

  • Monitoring RFX2-RASSF1-Hippo pathway activation might predict treatment response

• Male infertility diagnostics:

  • RFX2's critical role in spermatogenesis suggests diagnostic applications

  • Antibody-based assays could help identify specific subtypes of infertility related to RFX2 dysfunction

  • Testicular biopsy screening with RFX2 antibodies might provide prognostic information

• Single-cell analysis integration:

  • Combining RFX2 antibodies with single-cell technologies would reveal cell-specific expression patterns

  • This approach could identify specialized cell subpopulations with unique RFX2 regulatory networks

  • Particularly valuable for heterogeneous tissues like testis or tumor microenvironments

• Multi-omics integration:

  • Coupling ChIP-Seq using RFX2 antibodies with other -omics approaches (RNA-Seq, ATAC-Seq)

  • This would generate comprehensive models of RFX2-mediated gene regulation

  • Such models could reveal new therapeutic targets in cancer or reproductive medicine

• Spatial transcriptomics applications:

  • Integrating RFX2 antibody staining with spatial transcriptomics techniques

  • This would map RFX2 protein localization alongside gene expression patterns

  • Valuable for understanding tissue-specific regulatory networks in development and disease

These directions represent significant opportunities for expanding our understanding of RFX2 biology and translating this knowledge into clinical applications.

How can researchers address current limitations in RFX2 antibody applications?

Current limitations in RFX2 antibody applications can be addressed through several strategic approaches:

• Specificity challenges:

  • Develop monoclonal antibodies with enhanced specificity

  • Implement rigorous validation protocols including genetic models and orthogonal methods

  • Generate antibodies against diverse RFX2 epitopes to ensure detection in various contexts

• Species cross-reactivity limitations:

  • Most current antibodies show limited cross-species reactivity

  • Design antibodies targeting highly conserved regions of RFX2

  • The immunogen sequence VMGEFNDLASLSLTLLDKDDMGDEQRGSEAGPDARSLGEPLVKRERSDPNHSLQGI shows 80% identity between human and mouse/rat

• Post-translational modification detection:

  • Develop modification-specific antibodies (phospho-RFX2, acetyl-RFX2, etc.)

  • These would enable investigation of RFX2 regulation through post-translational mechanisms

  • Particularly relevant for understanding dynamic RFX2 activity in disease processes

• Technical improvements:

  • Optimize antibody formats for emerging technologies (e.g., super-resolution microscopy)

  • Develop antibody-based proximity labeling approaches for RFX2 interaction mapping

  • Create reversible immunoprecipitation systems for native complex isolation

• Integration with advanced technologies:

  • Apply RFdiffusion and related antibody design technologies to RFX2

  • Use AlphaFold3-like modeling to predict optimal antibody-RFX2 interactions

  • Develop multiplexed detection systems for simultaneous analysis of RFX2 and interacting partners

• Standardization efforts:

  • Establish community-wide validation standards for RFX2 antibodies

  • Create shared resources of validated positive and negative control samples

  • Develop quantitative benchmarks for antibody performance across applications

These approaches would significantly enhance the utility and reliability of RFX2 antibodies in research settings.

What clinical applications might emerge from advanced RFX2 antibody research?

Advanced RFX2 antibody research could enable several promising clinical applications:

• Cancer diagnostics and prognostics:

  • RFX2 expression is markedly downregulated in LUAD compared to normal tissue

  • Standardized IHC protocols using optimized RFX2 antibodies could serve as biomarkers

  • Potential applications in:

    • Distinguishing LUAD from other lung cancer subtypes

    • Predicting immune infiltration patterns and immunotherapy response

    • Monitoring disease progression through RFX2 pathway activation

• Reproductive medicine applications:

  • Male RFX2-deficient mice are completely sterile due to developmental arrest of haploid cells

  • Testicular biopsy analysis with RFX2 antibodies could:

    • Identify specific molecular causes of infertility

    • Guide personalized treatment approaches

    • Predict success rates for assisted reproductive technologies

• Therapeutic monitoring:

  • As therapeutics targeting the RFX2-RASSF1-Hippo pathway emerge, antibodies could:

    • Monitor treatment efficacy through pathway activation markers

    • Identify resistance mechanisms

    • Guide combination therapy approaches

• Companion diagnostics:

  • RFX2 expression correlates with CD8+ T cell infiltration in LUAD

  • This relationship suggests potential as a companion diagnostic for immunotherapy

  • RFX2 IHC might help stratify patients for clinical trials or standard-of-care immunotherapies

• Liquid biopsy development:

  • Modified RFX2 antibodies could potentially capture circulating tumor cells expressing RFX2

  • This approach might enable minimally invasive monitoring of LUAD

  • Sequential monitoring could track disease evolution and treatment response